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Basal lamina persistence during epithelial-mesenchymal interactions in murine tooth development in vitro.

Numerous investigations have demonstrated the necessity of mesenchymal instruction for epithelial differentiation during epidermal organogenesis. In the specific case of tooth formation, cap-stage tooth organ mesenchyme instructs epithelial differentiation into ameloblasts with production of enamel extracellular matrix. The "instructive event" is presumed to be direct cell contact. Mesenchyme-mediated cell contact with adjacent epithelia is assumed to "instruct" epithelial differentiation into ameloblasts. If this were true, basal lamina removal and mesenchyme cell contact with epithelia would be prerequisites for epithelial cytodifferentiation and morphogenesis in the developing tooth system. To test this hypothesis, we designed experiments to evaluate basal lamina stability during epithelial differentiation into ameloblasts. Our studies utilized cap-stage murine molar tooth organs, a serumless and chemically defined medium (PYMS), metabolic isotopic labeling of basal lamina constituents, biochemical methods to analyze macromolecular stability throughout 10 days of organ culture in vitro, and immunological methods to localize the distribution of laminin and fibronectin. Our results indicate that (3H)glucosamine is incorporated into basement membranes present in Theiler stage 25 mandibular mouse molar tooth organ. At this stage, the isotope was incorporated into high molecular weight macromolecules. Specific enzyme methods coupled with electrophoresis and fluorography demonstrated that (3H)glucosamine was incorporated into proteoglycans containing chondroitin sulfates, dermatan sulfate, and hyaluronate. After 10 days in vitro the radiolabeled material remained localized in these same molecules, indicating stability of these constituents within basement membranes. Ultrastructural observations indicated that the basal lamina was not removed during ameloblast differentiation in vitro using PYMS medium. Laminin and fibronectin were localized in the basement membranes during cap stages and did not disappear during subsequent morphogenesis and differentiation. Mesenchymal cells appear to mediate epithelial differentiation in vitro using PYMS medium without a removal of the basal lamina.

Animals↗

How to analyze and understand the human immune system.

To enhance our understanding of the pathogenesis of diseases, including rheumatic diseases, and to improve disease control, it is essential to attain a thorough understanding of the human immune system, alongside mouse immunology. Historically, the investigation of the human immune system has posed significant challenges due to methodological limitations. Nonetheless, recent advancements in genomic studies of multifactorial diseases have elucidated that numerous risk-associated genetic variants affecting quantitative differences in cell-specific gene expression. In light of these findings, we are currently examining individual genetic variations in both healthy individuals and patients, as well as categorizing cells into distinct subsets in order to construct a comprehensive dataset concerning the human immune system. This is accomplished by combining data on gene expression, factors influencing the expression mechanisms, protein expression, metabolomics, and environmental variables pertinent to immune functionality-such as gut microbiota. These datasets will facilitate the comprehensive characterization of the human immune system. Using these datasets and through the integrative analyses of data related to risk genetic variations and gene expression profiles of each disease and individual, we anticipate uncovering novel insights into the human immune system, the heterogeneity of diseases, immune function mechanisms, and their regulatory strategies that may not be achievable through murine models.

Humans↗

Group B streptococcal capsular sialic acids interact with siglecs (immunoglobulin-like lectins) on human leukocytes.

Group B Streptococcus (GBS) is classified into nine serotypes that vary in capsular polysaccharide (CPS) architecture but share in common the presence of a terminal sialic acid (Sia) residue. This position and linkage of GBS Sia closely resembles that of cell surface glycans found abundantly on human cells. CD33-related Siglecs (CD33rSiglecs) are a family of Sia-binding lectins expressed on host leukocytes that engage host Sia-capped glycans and send signals that dampen inflammatory gene activation. We hypothesized that GBS evolved to display CPS Sia as a form of molecular mimicry limiting the activation of an effective innate immune response. In this study, we applied a panel of immunologic and cell-based assays to demonstrate that GBS of several serotypes interacts in a Sia- and serotype-specific manner with certain human CD33rSiglecs, including hSiglec-9 and hSiglec-5 expressed on neutrophils and monocytes. Modification of GBS CPS Sia by O acetylation has recently been recognized, and we further show that the degree of O acetylation can markedly affect the interaction between GBS and hSiglec-5, -7, and -9. Thus, production of Sia-capped bacterial polysaccharide capsules that mimic human cell surface glycans in order to engage CD33rSiglecs may be an example of a previously unrecognized bacterial mechanism of leukocyte manipulation.

Animals↗

A virulent nonencapsulated Haemophilus influenzae.

Nontypeable Haemophilus influenzae strain INT1 was isolated from the blood of a young child with clinical signs of meningitis following acute otitis media. No immunologic or anatomic predisposition of this child for invasive bacterial infection with an unusual organism was documented. Sensitive ELISA proved the absence of intra- or extracellular capsular polysaccharide production by INT1 and Southern blot analysis confirmed the lack of an intact capsulation (cap) gene locus within the chromosome. Nevertheless, INT1 established bacteremia and meningitis in infant and weanling rat models of invasive H. influenzae infection. High-molecular-weight DNA isolated from INT1 was shown to confer an invasive phenotype on transformation of a nonencapsulated, avirulent laboratory strain of H. influenzae. Together these findings imply the presence of one or more as-yet-undiscovered, noncapsular virulence factors of H. influenzae that are capable of mediating invasive disease and resistance to immunologic clearance.

Amoxicillin↗

Giant liposomes as model membranes for immunological studies: spontaneous insertion of purified K1-antigen (poly-alpha-2,8-NeuAc) of Escherichia coli.

A flow chamber has been constructed to use giant liposomes (diameter 5-50 microns) as model membranes for immunological studies and other experiments involving the interaction with water-soluble compounds. As an example of immunological importance, the insertion of purified K-antigen from Escherichia coli K1 has been studied. Despite its large hydrophilic part (poly-alpha-2,8-NeuAc), which is capped at its potential reducing end with phosphatidic acid acting as a lipid anchor group, this water-soluble material is readily incorporated into liposomal membranes of dimyristoylphosphatidylcholine (DMPC). The incorporation has been proven by immunofluorescence using a FITC-labeled monoclonal anti-K1-IgG. Without the lipid residue, however, no binding of poly-alpha-2,8-NeuAc to the liposomes has been observed. This could be shown by using colominic acid, an oligomeric form of alpha-2,8-NeuAc with free reducing ends instead of purified K1-antigen. The possibility for further manipulation of this model system has been shown by using a poly-alpha-2,8-NeuAc cleaving enzyme (endoneuraminidase). The function of the endoneuraminidase has been proven by showing no binding of the antibody after enzyme treatment of K1-bearing liposomes as well as by rapid loss of fluorescence of a previously bound FITC-antibody.

Antibodies, Monoclonal↗

[Latex allergy--report on two cases].

In the last few years latex allergy has been increasingly recognised as a potential medical problem because of the increase in frequency and potential severity of latex-mediated reactions. Latex allergy is an IgE-mediated hypersensitivity response to natural rubber latex protein with a variety of clinical signs ranging from contact urticaria, angioedema, asthma, and anaphylaxis. Also IV. type of immunology response can participate in it. In highest risk for latex allergy are patients with spina bifida, but health care workers, latex industry workers, patients with multiple surgical procedures and others who wear gloves are also at risk. Patients with history of atopy belong to the high risk group. Diagnosis is done by positive in vitro tests (EAST, CAP-FEIA, immunoblott etc.) and skin prick test. Allergen avoidance and substitution and the use of latex-safe devices including synthetic gloves are essential for the affected patient.

Adult↗

Cutaneous vasculitis and reactive arthritis following respiratory infection due to Chlamydia pneumoniae: report of a case.

Unlike Chlamydia trachomatis and C. psittaci, the association of C. pneumoniae infection with immunological complications, such as reactive arthritis (ReA) or erythema nodosum (EN) has been rarely reported. Here we present the case history of a patient with C. pneumoniae community acquired pneumonia (CAP) who subsequently developed a ReA and a cutaneous vasculitis. A 45-year-old HLA B27 negative male developed an asymmetric and additive arthritis and a cutaneous leukocytoclastic vasculitis with IgM and complement papillary deposition along hypodermic vessel walls about three weeks after the onset of respiratory symptoms. The diagnosis of chronic Chlamydia pneumoniae infection was based on serology and PCR. Cultural and serological investigations for other infectious agents commonly involved in ReA were negative. This is the first report on the occurrence of two immune-based complications, associated to Chlamydia pneumoniae infection. Therefore, since this infection is very common in our population, although often asymptomatic, should be systematically considered as a common causative agent of ReA and of vasculitis.

Arthritis, Reactive↗

Immunolocalization of an annexin-like protein in corn.

Although calcium has been proposed to be an important regulatory element in plant gravitropic growth, as yet no specific function of Ca2+ in growth regulation has been discovered. Our recent studies on a Ca(2+)-binding protein in pea seedlings called p35 indicate that it is a member of the annexin family of proteins and may play a key role in growth regulation through its function in delivering polysaccharides needed for wall construction. We previously reported the isolation of p35 from pea plumules and the production of polyclonal antibodies to it. Immunolocalizaton analyses of p35 in pea tissues revealed high levels of staining in secretory cell types such as developing vascular cells and outer root cap cells. To test how general was the occurrence and distribution of this annexin-like protein in plant cells we initiated an analysis of annexins in the monocot corn using immunological techniques. Our results indicate the immunochemical properties and localization of corn annexins are very similar to those reported for pea. They are consistent with the postulate that annexins may play a general role in the regulation of the secretion of wall polysaccharides needed for growth, and thus could be an important target of calcium action during gravitropic growth.

Annexins↗

Translation of homologous and heterologous messenger RNAs in a yeast cell-free system.

A stable mRNA-dependent cell-free translation system from Saccharomyces cerevisiae, prepared by a modification of the method of Hofbauer et al. [Eur. J. Biochem. 122 (1982) 199-203] was active in translation of exogenous homologous and heterologous mRNAs. Optimal translational activity required the addition of polyamines and yeast tRNA. The m transcript of the M segment of double-stranded RNA, synthesized in vitro using the killer virus-associated RNA polymerase, directed the synthesis of preprotoxin polypeptide (M-p32), which was immunologically identified using antitoxin antibody. Sindbis virus capsid protein and rabbit globin were also translated from their mRNAs. Translation was inhibited by puromycin, sparsomycin and anisomycin. Analogues of the 5'-terminal caps present on most eukaryotic mRNA molecules inhibited translation of added mRNAs, including capped mRNAs and the uncapped killer virus mRNA.

Cell-Free System↗

Vein replacement with fresh vital veins: a comparison of transplantation in RT-1-different rat strain combinations.

Transplantation of the intrathoracic inferior vena cava into the abdominal inferior vena cava of the recipient was performed in inbred rat strains of the following combinations: Syngeneic F344 leads to F344, RT-1-identical allogeneic LEW leads to F344, and RT-1-different allogeneic CAP leads to F344. In the RT-1-different combination subgroups with presensitization (blood, skin) were formed. The transplanted veins were evaluated at different times, macro- and microscopically. To test humoral immunology reactivity, we used a modified hemagglutination test, to analyse cell-mediated reactivity, subsequent skin grafts. The following results were obtained: 1) Aside from sporadic mononuclear cells caused by the operation technique, syngeneic vein grafts do not show any macroscopically visible and microscopically detectable changes. 2) Weakly allogeneic vein grafts do not lead to microscopically detectable changes in the graft. Strongly allogeneic vein grafts cause temporary mononuclear cell infiltration, which subsides within 21 days, without influencing the morphologic structure of the vessel wall. 3) In the RT-1-identical allogeneic strain combination, only accelerated skin graft rejection time indicates systemic sensitization, in the strongly allogeneic system, the increase of hemagglutinating antibodies also does this. 4) In both the RT-1-identical allogeneic strain combination and the RT-1-different allogeneic one, a proven sensitization does not lead to a permanent morphologic alteration. 5) Only presensitization with strong antigens (skin) leads to a macroscopically visible, as well as microscopically detectable, rejection reaction.

Animals↗

Disseminated intravascular coagulation in catastrophic antiphospholipid syndrome: clinical and haematological characteristics of 23 patients.

BACKGROUND: Disseminated intravascular coagulation (DIC) is an acquired syndrome characterised by formation of microthrombi and fibrin deposition in the microvasculature. The catastrophic antiphospholipid syndrome (APS) is characterised by multiorgan thrombosis, mainly involving small vessels. A broad spectrum of disorders may develop DIC features; however, the catastrophic APS has not previously been recognised as a cause of DIC. OBJECTIVE: To analyse the clinical and laboratory characteristics of catastrophic APS patients with DIC features. METHODS: The web site based international registry of patients with catastrophic APS (CAPS registry) (http://www.med.ub.es/MIMMUN/FORUM/CAPS.HTM) was analysed and the cases with DIC features selected. RESULTS: In 173 patients with catastrophic APS, 23 (13%) were found with DIC features. The clinical and immunological characteristics were similar in catastrophic APS patients with and without DIC features; a significant difference was found only in the prevalence of thrombocytopenia (100% in patients with DIC features v 59% in those without DIC features). CONCLUSIONS: DIC features are not rare in catastrophic APS, supporting the need for systematic screening of antiphospholipid antibodies in all patients with DIC features without precipitating factors. The presence of DIC features in the context of an APS makes it imperative to rule out the catastrophic variant of this syndrome.

Adolescent↗

Stabilization of an isolated helical capping box in solution by hydrophobic interactions: evidence from the NMR study of bioactive peptides from the C-terminus of human C5a anaphylatoxin.

Synthetic analogues of the C-terminal portion of C5a were designed and found to be agonists of the C5a receptor [J. A. Ember et al. (1992) Jounral of Immunology, Vol. 148, p. 3165]. Nuclear magnetic resonance experiments were carried out to determine the solution conformation of the most potent analogue, the peptide C5a 65-74 (Tyr65, Phe67) (Tyr65-Ser66-Phe67-Lys68-Asp69-Met70 -Gln71- Leu72-Gly73-Arg74). Medium-range nuclear Overhauser effects (NOEs) were observed for residues 65-70 of this C5a peptide, suggesting that this region adopts a folded conformation in a significant population of the solution conformational ensemble. Quantitative analyses of (3)J(NH-alphaH) coupling constants and sequential NOE cross peaks gave an estimated helical population of 65% in the region Ser66-Met70. Additional evidence supporting the presence of a helical turn includes reduced amide-proton temperature coefficients and lowered (3)J(NH-alphaH) coupling constants in the region of Phe67-Met70. Conformational behavior of this C5a analogue peptide was studied using molecular modeling incorporating observed NOEs as constraints. The side chains of Tyr65, Phe67, and Met70 consistently form a hydrophobic cluster in all the model structures. The side chains of residues Ser66 and Asp69 can form reciprocal hydrogen bonds with the backbone NH groups of these two residues, indicating that residues Ser66-Phe67-Lys68-Asp69 (or SFKD) form a helix-stabilizing capping box (E. T. Harper and G. D. Rose (1993) Biochemistry, Vol. 32, p. 7605; H. X. Zhou et al. (1994) Proteins: Structure, Function and Genetics, Vol. 18, p. 1] even within the single turn of helical structure found in the analogue C5a peptide. A comparison of nmr results obtained for the analogue peptide and the natural decapeptide C5a 65-74 (Ile65-Ser66-His67-Lys68-Asp-69- Met70-Gln71-Leu72-Gly73-Arg74) indicated that incorporation of residues Tyr65 and Phe67 helps stabilize an isolated capping box involving residues Ser66-Asp69 in the C5a peptides through more extensive hydrophobic/aromatic interactions between residues Tyr65, Phe67, and Met70 in the analogue peptide C5a 65-74 (Tyr65, Phe67). These results constitute the first experimental demonstration of hydrophobic determinants in helical capping-box interactions, proposed recently by a statistical analysis of protein structures [J. W. Seale et al. (1994) Protein Science, Vol. 3, pp. 1741-1745]. The stabilized helical turn may also account for the greater potency of the analogue peptide C5a65-74 (Tyr65, Phe67) in receptor-binding assays.

Amino Acid Sequence↗

Lens-induced uveitis: further immunological studies in an experimental model.

Inbred rats have been studied to determine under what circumstances sensitization will or will not induce uveitis. After 50 days of biomicroscopic follow-up and histologic evaluation of 119 eyes the following was found in black hooded CAP-rats (RtH-1c) presensitized to eye, liver or kidney (guinea pig): 1. Alterations only following additional damage to their own tissue by lens discission plus iridotomy 2. As the case of lens-induced uveitis in humans, alterations after the disappearance of traumatization reaction in the form of protein-rich exudate clinically and an accumulation of round cells in the anterior uvea, surrounding the lens and retinal vessels histologically 3. Dependence of these alterations on sensitization by xenogenic tissue 4. Dependence of the alterations on the animal strain, i.e. absence in albino LEW (RtH-1(1)).

Animals↗

Solution and solid-support synthesis of a potential leishmaniasis carbohydrate vaccine.

The synthesis of a potential carbohydrate vaccine for the parasitic disease leishmaniasis is described. New solution- and solid-phase synthetic strategies were explored for the assembly of a unique tetrasaccharide antigen found on the Leishmania lipophosphoglycan. An initial solution-phase synthesis relied on thioglycosides as building blocks and the establishment of the central disaccharide from lactal via an oxidation-reduction sequence. A second approach was completed both in solution and on solid support. The solid-phase synthesis relied on assembly from monosaccharide units and was used to evaluate different glycosylating agents in the efficient installation of the galactose beta-(1-->4) mannoside. Glycosyl phosphates proved most successful in this endeavor. This first solid-phase synthesis of the Leishmania cap provided rapid access to the tetrasaccharide in 18% overall yield while requiring only a single purification step. The synthetic cap tetrasaccharide was conjugated to the immunostimulator Pam3Cys to create fully synthetic carbohydrate vaccine 1 and to the carrier protein KLH to form semisynthetic vaccine 2. Currently, both constructs have entered initial immunological experiments in mice targeted at the development of a vaccine against the parasitic disease leishmaniasis.

Animals↗